Marta Campiglio

The role of auxiliary subunits for the functional diversity of voltage-gated calcium channels.

Voltage‐gated calcium channels (VGCCs) represent the sole mechanism to convert membrane depolarization into cellular functions like secretion, contraction, or gene regulation. VGCCs consist of a pore‐forming α1 subunit and several auxiliary channel subunits. These subunits come in multiple isoforms and splice‐variants giving rise to a stunning molecular diversity of possible subunit combinations. It is generally believed that specific auxiliary subunits differentially regulate the channels and thereby contribute to the great functional diversity of VGCCs. If auxiliary subunits can associate and dissociate from pre‐existing channel complexes, this would allow dynamic regulation of channel properties. However, most auxiliary subunits modulate current properties very similarly, and proof that any cellular calcium channel function is indeed modulated by the physiological exchange of auxiliary subunits is still lacking. In this review we summarize available information supporting a differential modulation of calcium channel functions by exchange of auxiliary subunits, as well as experimental evidence in support of alternative functions of the auxiliary subunits. At the heart of the discussion is the concept that, in their native environment, VGCCs function in the context of macromolecular signaling complexes and that the auxiliary subunits help to orchestrate the diverse protein–protein interactions found in these calcium channel signalosomes. Thus, in addition to a putative differential modulation of current properties, differential subcellular targeting properties and differential protein–protein interactions of the auxiliary subunits may explain the need for their vast molecular diversity. J. Cell. Physiol. 999: 00–00, 2015.

Stable incorporation versus dynamic exchange of β subunits in a native Ca2+ channel complex

Summary Voltage-gated Ca2+ channels are multi-subunit membrane proteins that transduce depolarization into cellular functions such as excitation–contraction coupling in muscle or neurotransmitter release in neurons. The auxiliary &bgr; subunits function in membrane targeting of the channel and modulation of its gating properties. However, whether &bgr; subunits can reversibly interact with, and thus differentially modulate, channels in the membrane is still unresolved. In the present study we applied fluorescence recovery after photobleaching (FRAP) of GFP-tagged &agr;1 and &bgr; subunits expressed in dysgenic myotubes to study the relative dynamics of these Ca2+ channel subunits for the first time in a native functional signaling complex. Identical fluorescence recovery rates of both subunits indicate stable interactions, distinct recovery rates indicate dynamic interactions. Whereas the skeletal muscle &bgr;1a isoform formed stable complexes with CaV1.1 and CaV1.2, the non-skeletal muscle &bgr;2a and &bgr;4b isoforms dynamically interacted with both &agr;1 subunits. Neither replacing the I–II loop of CaV1.1 with that of CaV2.1, nor deletions in the proximal I–II loop, known to change the orientation of &bgr; relative to the &agr;1 subunit, altered the specific dynamic properties of the &bgr; subunits. In contrast, a single residue substitution in the &agr; interaction pocket of &bgr;1aM293A increased the FRAP rate threefold. Taken together, these findings indicate that in skeletal muscle triads the homologous &bgr;1a subunit forms a stable complex, whereas the heterologous &bgr;2a and &bgr;4b subunits form dynamic complexes with the Ca2+ channel. The distinct binding properties are not determined by differences in the I–II loop sequences of the &agr;1 subunits, but are intrinsic properties of the &bgr; subunit isoforms.

Splice variants of the CaV1.3 L-type calcium channel regulate dendritic spine morphology.

Dendritic spines are the postsynaptic compartments of glutamatergic synapses in the brain. Their number and shape are subject to change in synaptic plasticity and neurological disorders including autism spectrum disorders and Parkinson’s disease. The L-type calcium channel CaV1.3 constitutes an important calcium entry pathway implicated in the regulation of spine morphology. Here we investigated the importance of full-length CaV1.3L and two C-terminally truncated splice variants (CaV1.342A and CaV1.343S) and their modulation by densin-180 and shank1b for the morphology of dendritic spines of cultured hippocampal neurons. Live-cell immunofluorescence and super-resolution microscopy of epitope-tagged CaV1.3L revealed its localization at the base-, neck-, and head-region of dendritic spines. Expression of the short splice variants or deletion of the C-terminal PDZ-binding motif in CaV1.3L induced aberrant dendritic spine elongation. Similar morphological alterations were induced by co-expression of densin-180 or shank1b with CaV1.3L and correlated with increased CaV1.3 currents and dendritic calcium signals in transfected neurons. Together, our findings suggest a key role of CaV1.3 in regulating dendritic spine structure. Under physiological conditions it may contribute to the structural plasticity of glutamatergic synapses. Conversely, altered regulation of CaV1.3 channels may provide an important mechanism in the development of postsynaptic aberrations associated with neurodegenerative disorders.

STAC3 stably interacts through its C1 domain with CaV1.1 in skeletal muscle triads

The adaptor protein STAC3 is essential for skeletal muscle excitation-contraction (EC) coupling and a mutation in the STAC3 gene has been linked to a severe muscle disease, Native American myopathy (NAM). However the function of STAC3, its interaction partner, and the mode of interaction within the EC-coupling complex remained elusive. Here we demonstrate that STAC3 forms a stable interaction with the voltage-sensor of EC-coupling, CaV1.1, and that this interaction depends on a hitherto unidentified protein-protein binding pocket in the C1 domain of STAC3. While the NAM mutation does not affect the stability of the STAC3-CaV1.1 interaction, mutation of two crucial residues in the C1 binding pocket increases the turnover of STAC3 in skeletal muscle triads. Thus, the C1 domain of STAC3 is responsible for its stable incorporation into the CaV1.1 complex, whereas the SH3 domain containing the NAM mutation site may be involved in low-affinity functional interactions in EC-coupling.

A Cav3.2/Stac1 molecular complex controls T-type channel expression at the plasma membrane

ABSTRACT Low-voltage-activated T-type calcium channels are essential contributors to neuronal physiology where they play complex yet fundamentally important roles in shaping intrinsic excitability of nerve cells and neurotransmission. Aberrant neuronal excitability caused by alteration of T-type channel expression has been linked to a number of neuronal disorders including epilepsy, sleep disturbance, autism, and painful chronic neuropathy. Hence, there is increased interest in identifying the cellular mechanisms and actors that underlie the trafficking of T-type channels in normal and pathological conditions. In the present study, we assessed the ability of Stac adaptor proteins to associate with and modulate surface expression of T-type channels. We report the existence of a Cav3.2/Stac1 molecular complex that relies on the binding of Stac1 to the amino-terminal region of the channel. This interaction potently modulates expression of the channel protein at the cell surface resulting in an increased T-type conductance. Altogether, our data establish Stac1 as an important modulator of T-type channel expression and provide new insights into the molecular mechanisms underlying the trafficking of T-type channels to the plasma membrane.

Structural insights into binding of STAC proteins to voltage-gated calcium channels

Significance Skeletal muscle contraction is a tightly orchestrated event that starts with the depolarization of the T-tubular membrane. At the center is a functional and mechanical coupling between two membrane proteins: L-type voltage-gated calcium channels, located in the plasma membrane, and ryanodine receptors, located in the membrane of the sarcoplasmic reticulum. How exactly these proteins associate has remained a mystery, but recent reports have highlighted a key role for the STAC3 adaptor protein in this process. Here, we provide structural snapshots of the three STAC isoforms and identify a cytosolic loop of two CaV isoforms as a functional interaction site. A mutation linked to Native American myopathy is at the interface and abolishes the interaction. Excitation–contraction (EC) coupling in skeletal muscle requires functional and mechanical coupling between L-type voltage-gated calcium channels (CaV1.1) and the ryanodine receptor (RyR1). Recently, STAC3 was identified as an essential protein for EC coupling and is part of a group of three proteins that can bind and modulate L-type voltage-gated calcium channels. Here, we report crystal structures of tandem-SH3 domains of different STAC isoforms up to 1.2-Å resolution. These form a rigid interaction through a conserved interdomain interface. We identify the linker connecting transmembrane repeats II and III in two different CaV isoforms as a binding site for the SH3 domains and report a crystal structure of the complex with the STAC2 isoform. The interaction site includes the location for a disease variant in STAC3 that has been linked to Native American myopathy (NAM). Introducing the mutation does not cause misfolding of the SH3 domains, but abolishes the interaction. Disruption of the interaction via mutations in the II–III loop perturbs skeletal muscle EC coupling, but preserves the ability of STAC3 to slow down inactivation of CaV1.2.

The juvenile myoclonic epilepsy mutant of the calcium channel β4 subunit displays normal nuclear targeting in nerve and muscle cells

Voltage-gated calcium channels regulate gene expression by controlling calcium entry through the plasma membrane and by direct interactions of channel fragments and auxiliary β subunits with promoters and the epigenetic machinery in the nucleus. Mutations of the calcium channel β4 subunit gene (CACNB4) cause juvenile myoclonic epilepsy in humans and ataxia and epileptic seizures in mice. Recently a model has been proposed according to which failed nuclear translocation of the truncated β4 subunit R482X mutation resulted in altered transcriptional regulation and consequently in neurological disease. Here we examined the nuclear targeting properties of the truncated β4b(1–481) subunit in tsA-201 cells, skeletal myotubes, and in hippocampal neurons. Contrary to expectation, nuclear targeting of β4b(1–481) was not reduced compared with full-length β4b in any one of the three cell systems. These findings oppose an essential role of the β4 distal C-terminus in nuclear targeting and challenge the idea that the nuclear function of calcium channel β4 subunits is critically involved in the etiology of epilepsy and ataxia in patients and mouse models with mutations in the CACNB4 gene.

STAC proteins associate to the IQ domain of CaV1.2 and inhibit calcium-dependent inactivation

Significance Association of the adaptor protein STAC3 to the calcium channel was recently identified as essential for skeletal muscle contraction. While STAC3 is expressed exclusively in skeletal muscle, the other two isoforms, STAC1 and STAC2, are found in the brain, but their function has not yet been investigated. Recently we identified the C1 domain of STAC as crucial for its association with calcium channels. However, the corresponding binding domain remained unknown. Here, we demonstrate that STAC proteins associate with the C terminus of CaV1.2 at the IQ domain, and that this interaction results in the inhibition of calcium-dependent inactivation. Taken together, our data identify a functionally important STAC interaction domain and suggest that STAC proteins modulate calcium entry through CaV1 channels. The adaptor proteins STAC1, STAC2, and STAC3 represent a newly identified family of regulators of voltage-gated calcium channel (CaV) trafficking and function. The skeletal muscle isoform STAC3 is essential for excitation–contraction coupling and its mutation causes severe muscle disease. Recently, two distinct molecular domains in STAC3 were identified, necessary for its functional interaction with CaV1.1: the C1 domain, which recruits STAC proteins to the calcium channel complex in skeletal muscle triads, and the SH3-1 domain, involved in excitation–contraction coupling. These interaction sites are conserved in the three STAC proteins. However, the molecular domain in CaV1 channels interacting with the STAC C1 domain and the possible role of this interaction in neuronal CaV1 channels remained unknown. Using CaV1.2/2.1 chimeras expressed in dysgenic (CaV1.1−/−) myotubes, we identified the amino acids 1,641–1,668 in the C terminus of CaV1.2 as necessary for association of STAC proteins. This sequence contains the IQ domain and alanine mutagenesis revealed that the amino acids important for STAC association overlap with those making contacts with the C-lobe of calcium-calmodulin (Ca/CaM) and mediating calcium-dependent inactivation of CaV1.2. Indeed, patch-clamp analysis demonstrated that coexpression of either one of the three STAC proteins with CaV1.2 opposed calcium-dependent inactivation, although to different degrees, and that substitution of the CaV1.2 IQ domain with that of CaV2.1, which does not interact with STAC, abolished this effect. These results suggest that STAC proteins associate with the CaV1.2 C terminus at the IQ domain and thus inhibit calcium-dependent feedback regulation of CaV1.2 currents.

Stapled Voltage-Gated Calcium Channel (CaV) α-Interaction Domain (AID) Peptides Act As Selective Protein–Protein Interaction Inhibitors of CaV Function

For many voltage-gated ion channels (VGICs), creation of a properly functioning ion channel requires the formation of specific protein–protein interactions between the transmembrane pore-forming subunits and cystoplasmic accessory subunits. Despite the importance of such protein–protein interactions in VGIC function and assembly, their potential as sites for VGIC modulator development has been largely overlooked. Here, we develop meta-xylyl (m-xylyl) stapled peptides that target a prototypic VGIC high affinity protein–protein interaction, the interaction between the voltage-gated calcium channel (CaV) pore-forming subunit α-interaction domain (AID) and cytoplasmic β-subunit (CaVβ). We show using circular dichroism spectroscopy, X-ray crystallography, and isothermal titration calorimetry that the m-xylyl staples enhance AID helix formation are structurally compatible with native-like AID:CaVβ interactions and reduce the entropic penalty associated with AID binding to CaVβ. Importantly, electrophysiological studies reveal that stapled AID peptides act as effective inhibitors of the CaVα1:CaVβ interaction that modulate CaV function in an CaVβ isoform-selective manner. Together, our studies provide a proof-of-concept demonstration of the use of protein–protein interaction inhibitors to control VGIC function and point to strategies for improved AID-based CaV modulator design.

Molecular mimicking of C-terminal phosphorylation tunes the surface dynamics of CaV1.2 calcium channels in hippocampal neurons

L-type voltage-gated CaV1.2 calcium channels (CaV1.2) are key regulators of neuronal excitability, synaptic plasticity, and excitation-transcription coupling. Surface-exposed CaV1.2 distributes in clusters along the dendrites of hippocampal neurons. A permanent exchange between stably clustered and laterally diffusive extra-clustered channels maintains steady-state levels of CaV1.2 at dendritic signaling domains. A dynamic equilibrium between anchored and diffusive receptors is a common feature among ion channels and is crucial to modulate signaling transduction. Despite the importance of this fine regulatory system, the molecular mechanisms underlying the surface dynamics of CaV1.2 are completely unexplored. Here, we examined the dynamic states of CaV1.2 depending on phosphorylation on Ser-1700 and Ser-1928 at the channel C terminus. Phosphorylation at these sites is strongly involved in CaV1.2-mediated nuclear factor of activated T cells (NFAT) signaling, long-term potentiation, and responsiveness to adrenergic stimulation. We engineered CaV1.2 constructs mimicking phosphorylation at Ser-1700 and Ser-1928 and analyzed their behavior at the membrane by immunolabeling protocols, fluorescence recovery after photobleaching, and single particle tracking. We found that the phosphomimetic S1928E variant increases the mobility of CaV1.2 without altering the steady-state maintenance of cluster in young neurons and favors channel stabilization later in differentiation. Instead, mimicking phosphorylation at Ser-1700 promoted the diffusive state of CaV1.2 irrespective of the differentiation stage. Together, these results reveal that phosphorylation could contribute to the establishment of channel anchoring mechanisms depending on the neuronal differentiation state. Finally, our findings suggest a novel mechanism by which phosphorylation at the C terminus regulates calcium signaling by tuning the content of CaV1.2 at signaling complexes.